Characterisation of 49 Bacillus cereus strains obtained from extended shelf life (ESL) milk and filler nozzles was done using (GTG) 5 Rep PCR fingerprinting; determining the presence of virulence genes; cytK, nheA, cer and hblA and discrimination of psychrotrophic and mesophilic strains with 16S rDNA. Fourteen isolates were selected for 16S partial sequencing. Fingerprinting and sequencing showed evidence of filler nozzles contaminating ESL milk despite high heteroginity existing between the isolates. While there is high prevalence of cer, hblA and nheA; cytK was not widely distributed. There was 100% and 8% prevalence of mesophilic and psychrotrophic signatures respectively. Despite the high diversity of the B. cereus in this study, there is evidence that filler nozzles and raw milk are a source of contamination of B. cereus in ESL milk.

We used rpoB partial sequencing and multilocus sequence typing (MLST) to characterize 7 Bacillus cereus strains obtained at the following points: ESL milk during shelf life, pasteurized milk, raw milk, and filler nozzles after cleaning in place. The objective of the study was to determine relatedness among B. cereus isolates from several sampling points along an ESL processing plant with the aim of source tracking. The study revealed that isolates from filler nozzles shared 100% similarity with isolates from ESL milk and raw milk using rpoB sequencing. It also revealed that isolates from pasteurized milk shared 100% similarities with isolates from filler nozzles and ESL milk using MLST. We suggest 3 routes of B. cereus contamination in ESL milk. We showed that B. cereus contamination of ESL milk might be through raw milk and biofilms from filler nozzles. In addition, rpoB partial sequencing and MLST can be used as tools for source tracking in ESL milk processing.

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log10 cfu/mL and 4 log10 cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.

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